Cloning, expression, and nucleotide sequence of the Escherichia coli K-12 ackA gene.

The Escherichia coli K-12 ackA gene, which encodes an acetate kinase, was cloned. The acetate kinase activities of ackA+ plasmid-containing strains were amplified 160- to 180-fold. The complete nucleotide sequence of the ackA gene was determined. It was deduced that the ackA gene coded for a protein of 400 amino acids with an Mr of 43,297. The ackA gene was found to be located about 15 kilobases upstream of the purF-folC-hisT region of the chromosome.     

Complete nucleotide sequence of the glutamate dehydrogenase gene from Escherichia coli K-12

A 2.3-kb PstI-ClaI chromosomal DNA segment, carrying the complete coding region of the glutamate dehydrogenase (GDH) structural gene from Escherichia coli K-12, has been sequenced. The complete amino acid sequence (447 residues) of the GDH monomer has been deduced, and comparisons are made with reported amino acid sequences of GDH from other organisms.     

The nucleotide sequence of recG, the distal spo operon gene in Escherichia coli K-12.

A gene is identified in the Escherichia coli K-12 spo operon as recG. Previously identified genes in the spo operon were spoS, alias rpoZ, encoding the omega (omega) subunit of RNA polymerase, as well as the spoT gene encoding the major cellular source of guanosine 3',5'-bispyrophosphate hydrolase activity. The gene order within the spo operon is: spoS (rpoZ), spoT, spoU, recG. A convergent gltS gene is present beyond the spo operon. Mutants bearing recG deletion-insertion alleles display mild sensitivities to both ultraviolet irradiation and to mitomycin C, which is expected to be due to a known recG insertion allele. Deletion-insertion mutations in upstream operon genes (spoT and spoU) show polar effects on these assays of recG function. The deduced 693-amino acid (aa) RecG sequence shows a weak, but significant, relatedness to aa sequence motifs previously reported for putative helicases involved in replication, recombination, and DNA repair.     

Peptidase D of Escherichia coli K-12, a metallopeptidase of low substrate specificity

Abstract Peptidase D of Escherichia coli was overproduced from a multicopy plasmid and purified to electrophoretic homogeneity. The pure enzyme was stable at 4°C or −20°C and had a pH optimum at pH 9, and a p I of 4.7; the temperature optimum was at 37°C. As the enzyme was activated by Co²⁺ and Zn²⁺, and deactivated by metal chelators, it appears to be a metallopeptidase. By activity staining of native gels, 11 dipeptides which are preferentially cleaved by peptidase D were identified. Peptidase D activity required dipeptide substrates with an unblocked amino terminus and the amino group in the α or β position. Non-protein amino acids and proline were not accepted in the C-terminal position, whereas some dipeptide amides and formyl amino acids were hydrolyzed. K _m values of 2 to 5 mM indicate a relatively poor interaction of the enzyme with its substrates.     

Isolation and nucleotide sequence analysis of tRNAAlaGGC from Escherichia coli K-12.

An alanine tRNA with the anticodon 5'-GGC-3' has been identified in Escherichia coli K-12. It is the first sequenced alanine tRNA with G in the 5' position of the anticodon. tRNAAlaGGC has A in the "semi-invariant" position 32. At the "invariant" position 8 we observed both U and another, unknown, nucleoside.     

Deletion mapping of the ilvGOEDAC genes of Escherichia coli K-12.

Summary A set of dilv phage has been examined that carry overlapping segments of isoleucine-valine structural and regulatory genes derived from the ilv cluster at 83 min on the Escherichia coli K-12 chromosome. The ilv genes present in these phage, and their order, have been determined by transduction of auxotrophs, escape synthesis, and deletion mapping. The order of ilv genes in the phage, and hence the order in the host chromosome, was found to be ilvG-ilvO-ilvEDA-ilvC. Lysogens containing dilv phage were constructed for dominance analysis of regulatory mutations in the ilvO and ilvA genes. The ilvO671 allele is cis-dominant to ilvO ⁺, while the ilvA538 allele is trans-recessive to ilvA ⁺. Thus, the ilvO gene, that is identified by cis-dominant regulatory mutations that result in increased ilvG and ilvEDA expression, is situated between and may be contiguous with ilvG and ilvEDA.     

Cloning, sequencing, and mapping of the bacterioferritin gene (bfr) of Escherichia coli K-12.

The bacterioferritin (BFR) of Escherichia coli K-12 is an iron-storage hemoprotein, previously identified as cytochrome b1. The bacterioferritin gene (bfr) has been cloned, sequenced, and located in the E. coli linkage map. Initially a gene fusion encoding a BFR-lambda hybrid protein (Mr 21,000) was detected by immunoscreening a lambda gene bank containing Sau3A restriction fragments of E. coli DNA. The bfr gene was mapped to 73 min (the str-spc region) in the physical map of the E. coli chromosome by probing Southern blots of restriction digests of E. coli DNA with a fragment of the bfr gene. The intact bfr gene was then subcloned from the corresponding lambda phage from the gene library of Kohara et al. (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987). The bfr gene comprises 474 base pairs and 158 amino acid codons (including the start codon), and it encodes a polypeptide having essentially the same size (Mr 18,495) and N-terminal sequence as the purified protein. A potential promoter sequence was detected in the 5' noncoding region, but it was not associated with an "iron box" sequence (i.e., a binding site for the iron-dependent Fur repressor protein). BFR was amplified to 14% of the total protein in a bfr plasmid-containing strain. An additional unidentified gene (gen-64), encoding a relatively basic 64-residue polypeptide and having the same polarity as bfr, was detected upstream of the bfr gene.     

DNA sequence of the Escherichia coli K-12 gamma-glutamyltranspeptidase gene, ggt.

The DNA sequence of ggt, the gene that codes for gamma-glutamyltranspeptidase (EC 2.3.2.2) of Escherichia coli K-12, has been determined. The sequence contains a single open reading frame encoding the signal peptide and large and small subunits, in that order. This result suggests that E. coli gamma-glutamyltranspeptidase is processed posttranslationally.     

Analysis of the ptsH-ptsI-crr region in Escherichia coli K-12: nucleotide sequence of the ptsH gene

The nucleotide sequence of an Escherichia coli DNA segment containing the ptsH gene and the first 162 nucleotides of the ptsI gene encoding, respectively, Hpr and enzyme I of the phosphoenolpyruvate-dependent glycose phosphotransferase system (PTS), was determined. The ptsH promoter was localized using the S1 mapping technique. A nucleotide sequence very similar to the consensus binding site for cAMP receptor protein was found in the -35 region of the ptsH promoter. The ptsH gene is transcribed in the same direction as the ptsI gene and the crr gene (encoding enzyme IIIGlc of the PTS). Analysis of the nucleotide sequence substantiates the notion that the ptsH-ptsI-crr genes constitute a polycistronic operon.     

The complete nucleotide sequence of the ilvGMEDA operon of Escherichia coli K-12.

In this report we present the complete nucleotide sequence of the ilvGMEDA operon of Escherichia coli. This operon contains five genes encoding four of the five enzymes required for the biosynthesis of isoleucine and valine. We identify and describe the coding regions for these five structural genes and the structural and functional features of the flanking and internal regulatory regions of this operon. This new information contributes to a more complete understanding of the overall control of the biosynthesis of isoleucine and valine.     

Sulfate and thiosulfate transport in Escherichia coli K-12: nucleotide sequence and expression of the cysTWAM gene cluster.

The nucleotide sequence of the sulfate and thiosulfate transport gene cluster has been determined and located 3' to the gene (cysP) encoding the thiosulfate-binding protein. Four open reading frames, designated cysT, cysW, cysA, and cysM, have been identified. Similarities in primary structure were observed between (i) the deduced amino acid sequences of CysT and CysW with membrane-bound components of other binding protein-dependent transport systems, (ii) that of the CysA sequence with the "conserved" component of such systems, and (iii) that of the CysM sequence with O-acetylserine sulfhydrylase A (cysK gene product) and the beta-subunit of tryptophan synthase (coded by trpB). Expression of the four genes was analyzed in the T7 promoter-polymerase system.